antiproliferative effects by lipocortins Partial mediation ...

Partial mediation of glucocorticoid antiproliferative effects by lipocortins This information is current as of March 10, 2010

WY Almawi, MS Saouda, AC Stevens, ML Lipman, CM Barth and TB Strom J. Immunol. 1996;157;5231-5239

References

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright ©1996 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Partial Mediation of Glucocorticoid Antiproliferative Effects by Lipocortins' Wassim Y. AImawi,** Myrna S. Saouda,* Anthony C. Stevens,t Claudia M. Barth,t and Terry B. Stromt

Mark 1. Lipman,+

G

lucocorticoids (GCS),~ as anti-inflammatory and immunosuppressive agents, are used in treating autoimmune diseases and graft rejection episodes (1, 2). However, despite their wide-spread use, the precise mechanism by which GCs mediate their antiproliferative effects remains evasive due in part to the myriad of biological effects mediated by the GCs. Among the mechanisms postulated for GC-mediated antiproliferative effects include blockade of activation-associated increases in transmembrane ionic fluxes ( 3 , 4), alteration in membrane lipid phospholipid profile (5, 6), inhibition of cytokine gene expression (7-9), andor induction of the calcium and phospholipid binding proteins, the lipocortins (10, 11). It is well documented that GCs induce lipocortin expression at the mRNA and protein levels. Lipocortins, due to their capacity to inhibit phospholipase A, (PLA,) activity (12, 13), block arachidonic acid (AA) release from membrane-bound stores, resulting in

*DepartmentofBiochemistry,Faculty of Medicine,AmericanUniversityof Beirut, Beirut, Lebanon; and 'Division of Clinical Immunology, Department of Medicine, Beth Israel Hospital and Harvard MedicalSchool, Boston, M A 02215 Received for publication November 30, 1995. Accepted for publicationSeptember 5, 1996. The costs of publication of this article were defrayed in part by the payment of in page charges. This article must therefore be hereby marked advertisement accordance with 18 U.S.C. Section 1 734 solely to indicate this fact.

the inhibition of PC and leukotriene (LT) production (14, 15). This prompted the conclusion that GCs exert their effects through lipocortin induction, which, in turn, blocks AA release and, consequently, PC and LT production (IO, 121, resulting in the suppression of select elements of the signal transduction pathway(s) ( I 3). Other reports challenged this conclusion by presenting data showing that GC-mediated suppression may be a separate event from classical lipocortin induction, assessed by the inhibition of eicosanoid production (15-17). This did not rule out a possible involvement of lipocortins in transducing GC-mediated effects via an, as yet, undisclosed mechanism (16, 18). Previously, we demonstrated that GC-mediated antiproliferative effects do not involve altering the generationof second messenger systems (rise in intracellular calcium, activation and translocation of protein kinase C from cytosolic to membrane-bound compartments) that operate as a consequence of cellular activation (19). GCs mediate their antiproliferative effects by inhibiting cytokine expression as 1) antiproliferative concentrations of dexamethasone (DEX) and prednisolone (PRED) blocked steady state IL-I (9), IL-2 (7),and IFN-y (8) mRNA expression; and 2) the combination of IL-1, IL-6, and IFN-y completely abrogated GC-mediated antiproliferative effects (7). Here we show that lipocortins mediate in part GC-mediated antiproliferative effects, indicating the existence of a lipocortin-dependent and independent pathways by which GCs mediate their effects.

' This work was supported by grants from the National Institutes of Health, the Kidney Foundation of Canada, and the American University of Beirut-M.P.P. Address correspondence and reprlnt requests to Dr. Wassim Y. Almawi, Department of Biochemistry, Faculty of Medicine, American University of Beirut, 850 Third Ave., New York, NY 10022-6222,

Materials and Methods

Abbreviations used in this paper: GCs, glucocorticoids; PLA,, phospholipase AL ,;TB, leukotriene B;, DEX, dexamethasone; PRED, prednisolone; PBML, perlpheral blood mononuclear lymphocytes; ETYA, eicosatetraynoic acid; NDGA, nordihydroguaiaretic acid; AA, arachldonic acid.

Venous blood from healthy volunteers was diluted 1/2 in saline, layered on Hypaque-Ficoll (S.G. 1.077, Pharmacia Fine Chemicals, Piscataway, NJ), and centrifuged for 20 min at 2000 X g. The interphase containing PBML was washed three times in saline and was resuspended at IO6 cells/ml in

Copyright 0 1996 by The American Association of Immunologists

Preparation of PBML

0022-1 767/96/$02.00


The glucocorticoids (GCs) dexamethasone (DEX) and prednisolone (PRED), i n a concentration-dependent fashion, profoundly inhibit mitogen-induced proliferation of human peripheral blood mononuclear lymphocytes (PBML). This inhibition was specific for GCs, as non-GC steroids were devoid of any antiproliferative capacity. GCs enhanced the mRNA (Northern blot) and protein (Western blot) expression of the calcium and phospholipid binding proteins lipocortin I, II, and V. As a consequence of mitogenic stimulation, PBML secrete PGE, and leukotriene B,(LTB,). Antiproliferative concentrations of both DEX and PRED as well as recombinant lipocortin I abolished PGE, and LTB, production, suggestingan involvement of lipocortins in GCmediated antiproliferative effects, possibly by inhibiting eicosanoid production and, consequently, mitogen-induced cellular proliferation. Whereas 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid mimicked DEX and PRED in inhibiting PGE, and LTB, production, neither 5,8,11,14-eicosatetraynoic acid nor nordihydroguaiaretic acid had any effect on mitogeninduced PBML proliferation, indicating that theGC-mediated antiproliferative effect is separate from theireffects on eicosanoid release. Furthermore, neutralizing anti-lipocortin I and anti-lipocortin II mAb, while reversing the inhibitory activity of DEX and PRED on PGE, and LTB, production, only partially reversed DEX- and PRED-mediatedantiproliferative effects. This indicates that theGC-mediated antiproliferative effect is not dependent on inhibition ofeicosanoid release by lipocortins and suggests the existence of lipocortin-dependent and lipocortin-independent pathways by which GCs mediate their antiproliferative effects. The Journal of Immunology, 1996, 157: 5231-5239.

5232

PARTIAL MEDIATION OF GLUCOCORTICOID EFFECTS BY LIPOCORTINS

RPMI 1640 culture medium (M. A. Bioproducts, Bethesda, MD) supplemented with 50 pM @ME (Sigma Chemical Co., St. Louis, MO), 2 mM L-glutamine (Life Technologies, Grand Island, NY), penicillin-streptomycin (Life Technologies) at 100 IU/ml and 100 pg/ml, respectively, and 10% (v/v) human type AB serum (M. A. Bioproducts), referred to hereafter as complete medium.

Reagents and Abs PMA and leupeptin were purchased from Sigma Chemical Co., and PHA was obtained from Difco Laboratories (Detroit, MI). Stock solutions of DEX, PRED, AA, 5,8,11,14-eicosatetraynoicacid (ETYA), nordihydroguiaretic acid (NDGA), cholesterol, /3-estradiol, and aldosterone were made in 70% ethanol and stored at -20°C until used (all obtained from Sigma Chemical Co.). All other steroids used in the study were supplied as oilbased injections from the Beth Israel Hospital Pharmacy (Boston, MA). Anti-lipoconin I (lA), 11, and V mAb were obtained from Biogen, Inc. (Cambridge, MA),courtesy of Dr. J. Browning. Gold-conjugated goat antimouse Abs were obtained from Bio-Rad (Mississauga, Ontario, Canada), and mouse IgGl was purchased from Ortho Pharmaceutical Corp. (Raritan, NJ).

Proliferation assays

Determination of LTB, and PGE, PBMC were cultured in 24-well, flat-bottom plates (Falcon, Lincoln Park, NJ) at 5 X lo5 cells/ml in completemedium. The cells were pretreated with the indicated agents for 4 h at 3 7 T , followed by stimulation with PHA plus PMA. Cellfree supernatants of cultured and treated PBML were assayed for LTB, and PGE by RIA using a commercially available kit from Amersham C o p (Arlington Heights, IL). All determinations were performed in duplicate.

Cellular fractionation PBML (lo6 cellslml) were stimulated with PHA (5 pg/ml) and PMA(5 ng/ml), cultured for 24 h at 37°C with or without test drugs, washed twice in HBSS (Life Technologies, Grand Island, NY), and resuspended atlo7cells/ml in extraction buffer (20 mM Tris-C1, pH7.2; 2 mM EDTA; 50 mM 2-ME; and 100 pg/ml leupeptin). The cells were then sonicated for 20 s and centrifuged at 50,000 X g for 1 h at 4°C. Cytosoliclipocortin-containingfractionswerelyophilizedatstoredat -20°C until assayed.

Western blot analysis Cytosolic lipocortin-containing fractions were subjected to 7.5% SDSPAGE according to the method of Laemmli (20). Proteins were transferred to nitrocellulose membranes, and the membranes were incubated with the primary Ab for 2 h atroom temperature. Gold-conjugated goat anti-mouse mAb was added to washed membranes, and the membranes were incubated for 18 to 20 h at room temperature.

Northern blot analysis Total cellular RNA was extracted by the guanidium isothiocyanateLiC1 method under strict RNase-free conditions (21). RNA (10 pgfiane) was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde (22) and electrotransferred onto Hybond N+ membranes (Amersham). The membranes were prehybridized at 42°C for 4 h in a prehybridization solution containing 50% (v/v) deionized formamide, l X Denhardt's solution, I % SDS, 1 mM NaCI, 5 mM Tris-CI (pH 7.4), and 10% dextran sulfate (Sigma Chemical Co.). [32P]dCTP-labeled (New England Nuclear) cDNA probes were then added, and the membranes were hybridized at 42°C for 12 to 18 h. After washing twice in 2 X SSC/O.l% SDS at 42°C and twice

FIGURE 1. Specificity of theGC-mediatedantiproliferative effect. The proliferation of PBML stimulated with PHA (5 pghnl) and PMA (5 ng/ml) and treated with culture medium (positive control), ethanol (vehicle control), or the indicated steroid at the indicated concentration. 72 hpost-cultureinitiation;control Proliferationwasdetermined valueswere:background cpm, 781 t 124; positive control cpm, 92,715 -+ 9,877; and ethanol control cpm, 86,552 2 9,140. Data as:(l - [(test pointsindicatethepercentsuppression,calculated - backgroundcpm)l) x cpm - backgroundcpm)/(controlcpm

100%

in 0.1 X SSC/O.l% SDS at 75°C for 20 min, and the membranes were exposed to Kodak x-ray film (Eastman Kodak, Rochester, NY) at -70°C for 24 to 72 h.

Results Comparative effects of GC and non-GC steroids on mitogeninduced T cell proliferation

The effect of GC and non-GC steroids on mitogen-induced PBML proliferation was assessed by adding the steroids (and ethanol), at lo-' to 10"o M, to PBML cultures stimulated with PHA ( 5 fig/ ml) and PMA ( 5 ng/ml; referred to thereafter as PHA-PMA) at culture initiation; proliferation was determined by measuring the cellular uptake of [3H]TdR 72 h postactivation. The results presented in Figure 1 show that, of all the steroids tested, only DEX (EC,, = 5 X lo-' M), betamethasone (EC,, = 7.5 X lo-' M), hydrocortisone (EC,, = 5 X 10" M), and PRED (EC,, = 5 X M) inhibited mitogen-induced cellular proliferation (EC,, = drug concentration yielding 50% suppression of PHA-PMA responses). In contrast, the non-GC steroids aldosterone, androsterone, cholesterol, diethylstilbesterol, /3-estradiol, nandrolone, pregnenolone, progesterone, and testosterone were devoid of antiproliferative capacity at all concentrations tested, as the proliferative responses of mitogen-stimulated PBML cultures treated with non-GC steroids were not statistically different from those of either positive or ethanol control cultures (data not shown). Suppression of mitogen- and alloantigen-induced T cell proliferation by GCs

The antiproliferative capacity of GCs was further investigated by adding ethanol, DEX, or PRED, at 10"' to IO" M, to PBML cultures stimulated with Con A (10 pg/ml; Fig. 2 . 4 ) or with mitomycin C-treated allogeneic cells (MLR; Fig. 2 B ) ; cellular proliferation was determined 3 days (Con A) or 5 days (MLR) postculture initiation. Both DEX and PRED, in a concentrationdependent fashion, inhibited Con A-induced and MLR-driven T


For mitogen-induced proliferation PBML (5 X IO5 cells/ml) were cultured in 96-well, flat-bottom microtiter plates (Nunc, Burlington, Ontario, Canada) and were stimulated with PHA (5 pg/ml) and PMA (5 ng/ml) or with Con A (10 pg/ml). The cells were incubated for 72 h at37°C in a 5% CO, humidified atmosphere. For MLR, PBML (lo6 cells/ml) were cocultured with mitomycin C-treated (25 pg/ml; SigmaChemical Co.) allogeneic cells (lo6 celldml)in complete medium containing 50 pM@ME (Sigma Chemical Co.) for 5 days at 37°C. [3H]TdR (1 pCi/well; New England Nuclear, Boston, MA) was added during the last 4 h of the culture period, and proliferation was determined by measuring the cellular uptake of [3H]TdR by liquid scintillation.

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The Journal of I m m u n o l o g y

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60 -

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DEX MP . .

10”O

1

Io

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10”

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Concentration (M)

cell proliferation (Fig. 2, A and B ) . Furthermore, DEX was 50- to 100-fold more potent than PRED in inhibiting both T cell proliferative responses, assessed by comparing the EC,, values for both agents. GCs induce lipocortin expression

The effect of GCs on lipocortin I, 11, and V expression was first assessed by examining the effect of DEX and PRED on lipocortin I and I1 steady state mRNA expression. Total cellular RNA, extracted from DEX-treated and mitogen-stimulated cultures, was subjected to Northern blot analysis using 32P-labeled lipocortin I and lipocortin I1 cDNA probes. PBML stimulation with PHA-PMA resulted in a reproducible induction of lipocortin I, but not lipocortin 11, mRNA expression. DEX, in a concentration-dependent fashion, up-regulated lipocortin I and lipocortin I1 mRNA expression, with the maximal re-

sponse seen at to 10” M (Fig. 3). Similarly, lipocortin I and I1 mRNA levels were up-regulated in PRED-treated and mitogenstimulated PBML cultures (data not shown). We next assessed whether GC-induced up-regulation in lipocortin I and lipocortin I1 steady state mRNA levels resulted in parallel increases in lipocortin I, lipocortin 11, and lipocortin V protein secretion. Cytosolic fractions of GC-treated and mitogen-stimulated PBML cultures were subjected to SDS-PAGEmestern blot analysis, using specific anti-lipocortin I, 11,and V mAbs. Parallel to its effect on lipocortin I mRNA, stimulation of PBML with PHA-PMA resulted in the induction of lipocortin I, but not lipocortin I1 or lipocortin V, protein secretion (Fig. 4, A and B ) . DEX (Fig. 4A) and PRED (Fig. 4B) in a concentration-dependent fashI, liion, enhancedthecytosolic accumulationoflipocortin pocortin 11, and lipocortin V proteins. Smaller amounts of lipocortin I and lipocortin I1 were also detected in the membrane


FIGURE 2. inhibition by GCs of mitogenA, and alloantigen-induced proliferation. PBML (5 X 1 O4 cells) were stimulated with Con A (1 o pg/mI); 6, PBML ( I .5 x 1 o5 cells) werestimulated with mitomycin C-treated allogeneic cells (1.5 x lo5 cells). Both groups ofcellswere treated with ethanol (vehiclecontrol), DEX, or PRED. Proliferation was assessed 3 days ( A ) or 5 days ( B ) postculture initiation. Proliferation values for Con A: background cpm, 1,123 5 258; positive control cpm, 108,849 ? 11,624; for MLR: background cpm, 51 3 2 126; positive control cpm, 74,083 rt 6,466. Data points are the mean of eight individually performed experiments and indicate the percent suppression calculated as follows: (1 - [(test cpm - backgroundcpm)/(control cpm background cpm)]) X 100%.

40


5234

-

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0

togen-stimulated PBML cultures were treated with the inhibitors ofAA release, ETYAandNDGA,and their supernatants were assayed for PGE, and LTB,. NDGA and ETYA, in a concentration-dependent manner, inhibited PGE, (Fig. 7A) and LTB, (Fig. 7B) production in a manneranalogoustoDEX,PRED,and lipocortin I. In contrast to DEX, which inhibited mitogen-induced proliferation at concentrations as low as M, NDGA and ETYA, at all concentrations tested, failed to inhibit mitogen-induced T cell proliferation (Fig. 8). To rule out the possibility that the GC-mediated antiproliferative effect was due to inhibition ofAA release, DEX-treated and mitogen-stimulated PBML cultures were reconstituted with exogenous AA, and proliferation was determined 72 h postincubation. Theresultspresented in Figure 9 demonstratethat,atallconcentrations tested, AA did not alter DEX-mediated antiproliferative effects (Fig. 9).

0

Effect of anti-lipocortin /-neutralizing mAb on DEX-induced inhibitionofeicosanoidproductionandcellularproliferation

1 8 S 4

LC I

-

PHA DEX (M)

‘

0

- + - -

+

+

10-5

10-6

+ 10-7

+ 10-8

FIGURE 3. Northern blot analysis of total cellular RNA. Total cellular R N A (10 & l a n e )was extracted from PBML stimulated with PHAPMA andtreatedwith DEX at to lo-’ M. Top, Ethidium bromide staining of total RNA electrophoresed on a 1% agarose denaturinggel.

Following Northern transfer, the membranes were probed with 32Plabeled lipocortin I (middle)and lipocortin II (bottom)cDNA and exposed to Kodak x-ray film for 24 h at -70°C.

fractions of GC-treated and mitogen-stimulated cultures (data not shown). Inhibition of eicosanoid production and Tcell proliferation by lipocortin I and GCs

We next investigated whether induction of lipocortins by PRED and DEX results in inhibition ofAA release and, subsequently, blockade of mitogen-induced cellular proliferation. Mitogen-stimulated cultures were treated with DEX, PRED, and lipocortin I, and their supernatants were assayed for PGE, and LTB, by RIA. DEX, PRED, and lipocortin I, in a concentration-dependent fashion, inhibited PGE, (Fig. 5 A ) and LTB, (Fig. 5B)production. The liaddition of 50 to 100 pM AA reversedDEX-,PRED-,and pocortin I-induced blockade of PGE, production, indicating that all three agents inhibit PGE, (and LTB,) production by blocking the release of AA (data not shown). We then assessed the effect of lipocortin I on mitogen-induced cellularproliferation by addinglipocortin I andDEX,at to M, to mitogen-stimulated cultures. Lipocortin I,in a concentration-dependent fashion, inhibited mitogen-induced T cell proliferation (Fig. 6). By comparison to lipocortin I, DEX was >15foldmorepotent in inhibiting T cellproliferation,assessed by comparing the EC,, values for both agents. Effects of NDGA and ETYA on cellular proliferation and eicosanoid production

To assess whether GC-induced antiproliferative effects was due to blockade of AA release (resulting from lipocortin induction),mi-

To determine whether DEX-induced inhibition of eicosanoid production and cellular proliferationwas mediated via lipocortins, the effects of neutralizing anti-lipocortin I and anti-lipocortin I1 mAb on DEX-mediated blockade of PGE, and LTB, production and on DEX-induced inhibition of mitogen-induced cellular proliferation were assessed. Anti-lipocortin I and anti-lipocortin I1 mAb, individually and in combination, totally abrogated DEX-induced inhibition ofPGE,andLTB,productionby mitogen-stimulated PBML cultures (Fig. 10). The addition of anti-lipocortin I mAb or anti-lipocortin I1 mAb to DEX-treated and PHA-PMA-stimulated cultures resultedin partial abrogation of DEX-induced inhibition of PBML proliferation, whichdidnotexceed45%of the control values (Fig. 11). Furthermore, the addition of both anti-lipocortin I and anti-lipocortin I1 mAb did not result in any additive or synergistic effect, hence demonstrating that the GC-mediated antiproliferative effect is partially mediated by lipocortins. Taken together, these results suggest that GC-mediated antiproliferative effects follow lipocortindependent and lipocortin-independent pathways via a mechanism distinct from the inhibition of AA release.

Discussion The GCs DEX and PRED, in a concentration-dependent fashion, profoundly inhibit the proliferation of human PBML cultures induced by mitogenic or allogeneic stimuli,and up-regulate lipocortin mRNA and protein expression. In view of the inducibility of lipocortins by GCs and the mimicry of GC effects by lipocortin I, we investigated whether GC-mediated antiproliferative effects are mediated by lipocortins. Insofar as GCs are known to suppress mitogen and Ag-induced cellular proliferation,we tested the capacityof non-GC steroids to inhibit mitogen-induced cellular proliferation. Immunosuppression was specific for the GCs, as non-GC steroids did not affect mitogen-elicited or OKT3-stimulated (7, 19)cellular proliferation. This is in contrast to published reports claiming that P-estradiol (23, 24), cholesterol (25), diethylstilbestrol(26), progesterone (27,28), testosterone (29). and other non-GC steroids (30, 31) are immunosuppressive. It should be noted that in the studies quoted, steroid-mediated immunosuppression was associated with conditions such as arthritis (23), cancer (26), pregnancy (27, 28), and adult thymectomy (29). predisposing factors that may have contributed to the decreased immunity reported. In contrast and similar to our finding, non-GC immunosuppression was either lacking (16,32) or seen only at toxic concentrations (33) in normal human subjects.


LC II-

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The Journal of Immunology

5235

F? IRRELEVENT 3' ANTIBODY

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LIPOCORTIN I1

LIPOCORTIN V FIGURE 4. Western blot analysis. Western blot analysis of cytoplasmic preparations of unstimulated PBML (UNSTIMULATED),of PBML stimulated with PHA-PMA (PHNPMA),and of PBML stimulated with PHA-PMA and treated with DEX ( A ) or with PRED ( B ) at the indicated concentrations. Membranes were hybridized with irrelevant lgGl or with anti-lipocortin I,It, and V Abs, and protein-antibody interactions were visualized by gold staining.

PHA-PMA stimulation was associated with a reproducible induction of lipocortin I, but not lipocortin 11, mRNA expression. Insofar as GCs up-regulate lipocortin mRNA expression at the transcriptional and post-transcriptional levels (13,34-38), here we demonstrate that antiproliferative concentrations of DEX and PRED up-regulate lipocortin mRNA and protein expression in mi-

togen-stimulated cultures. DEX and PRED, in a concentrationdependent fashion, up-regulated lipocortin I and induced lipocortin I1 steady state mRNA expression, with maximal effects seen at IO-" M. The decline in mRNA expression seen at concentrations higher than IO" M is most likely due to mRNA degradation, as previously suggested (IO, 39).


PHA+PMA PREDNISOLONE CONCENTRATION (M)

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PGE Production

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FIGURE 6 . Inhibition by DEX andlipocortin 1 of mitogen-induced PBML proliferation. PBML were stimulated with PHA-PMA and treated with DEX (closed triangles) or lipocortin 1 (LC I; open tri-

LTB Production

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FIGURE 5. Inhibition of eicosanoid production byDEX,PRED, and lipocortin I. PGE, ( A ) and LTB, (5) levels were determined by RIA in the culture supernatants of PBML stimulated with PHA-PMA and treated with DEX (circles),lipocortin 1 (squares),and PRED (triangles). Data points represent the mean of five individually performed experiments, and indicate suppression, calculated as: (1 - [(test conc. background conc.)/(controlconc. - background conc.)]) X 100%.

DEX and PRED also enhanced the cytosolic accumulation of lipocortin I, lipococortin 11, and lipocortin V proteins. Similar to earlier reports, lipocortins I and I1 migrated on SDS-PAGE as two bands with apparent molecular masses of 37 and 33 kDa, respectively (36,40). In our hands, a third band migrating at 31 kDa was consistently observed only in DEX-treated cultures; the nature and significance of this band have yet to be determined. Lipocortin I shares with DEX and PRED the capacity to block PGE, and LTB, synthesis and to inhibit mitogen-induced cellular proliferation. In view of the inducibility of lipocortins by GCs (34, 36), and the effect of lipocortins as PLA, inhibitors on blocking AA release and subsequently PG and LT synthesis (13, 41), we tested whether the GC-mediated antiproliferative effect is due to the induction of lipocortin expression, which, in turn, is associated with inhibition of AA release and metabolism. While the PG and LT inhibitors NDGA and ETYA (42, 43) share with GCs and lipocortin I the capacity to inhibit PGE, and LTB, production, both NDGA and ETYA failed to inhibit mitogen-induced cellular proliferation, indicating that GC- and lipocortin 1-mediated antiproliferative effects were not the result of inhibition of AA release and metabolism. Lipocortin I was reported to mediate several GC effects, including superoxide generation by A23 187-stimulated macrophages

(44), inhibition of IL-I-induced neutrophil migration (43, inhibition of cellular proliferation (37), and induction of cellular differentiation (36, 46). Here we showed that lipocortin I also inhibits mitogen-induced and OKT3-stimulated (7, 19) cellular proliferation. Our results clearly demonstrate that GC-mediated inhibition of eicosanoid production was the result of induction of lipocortins, as has also been reported in bronchial alveolar lavage cells (47), in A549 lung adenocarcinoma cells (37, 48), and in differentiated U-937 cells (34). However, GC-mediated antiproliferative effects were not exclusively due to enhanced lipocortin expression, since anti-lipocortin I and anti-lipocortin II mAb, while totally abrogating the GC-mediated inhibition of eicosanoid production, only partially antagonizedGC-mediated antiproliferative effects. In essence,the GC-mediated inhibition of eicosanoid production appears to be lipocortin dependent (47, 48), while the antiproliferative effects follow both lipocortin-dependent and lipocortin-independent pathways (48, 49). The role of lipocortins in GC-mediated antiproliferative effects remains to be determined. Lipocortins, which are phosphorylated on tyrosine, serine, and threonine residues by src-like kinases (5052), may block the action of certain elements in the signaling pathway that operate as a consequence of cellular stimulation, hence leading to decreased cellular proliferation. In addition to partially acting via lipocortins, GCs may exert their antiproliferative effects directly by binding their cytosolic receptor, which, when translocating to the nucleus, binds the promoter region of several cytohne genes on specific sites collectively referred to as GC response elements (53-55). Binding of GC receptor complex to GC response element DNA sites inhibits cellular proliferation through blockade of cytokine gene expression at the transcriptional and transcriptional levels in cis- or trans-acting fashions, as previously reported (54, 56). In conclusion, the demonstration that lipocortins, while mediating GC effects in inhibiting the release of AA release and eicosanoid production, only partially mediate GC-associated antiproliferative effects is in line with our earlier thesis (7, 19, 53) that


angles). Proliferationwas assessed 3 days after culture initiation; control cpm values were: background cpm, 404 ? 103; positive control cpm, 82,679 f 8,629; and ethanol control cpm, 69,844 ? 8,379. Data points are the mean of six individually performed experiments and indicate the percent suppression, calculated as described in Fig. 2.

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FIGURE 7. Inhibitionofeicosanoidproduction by NDCA and ETYA.RIA of PCE, ( A ) and LTB, ( 6 )levels in the culture supernatants of PBML stimulated with PHA-PMA and treated with NDGA (closedcircles)or ETYA (opencircles).Datapoints represent the mean of five individually performed experiments and indicate the percent suppression, calculated as described in Figure 5.

A


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FIGURE 8. Failure of NDCA and ETYA to inhibit mitogen-induced proliferation. PBML were stimulated with PHA-PMA and treated with DEX (solid squares),ETYA (open squares), or NDCA (open circles). Data points represent the mean of four individually performed experiments; control cpm values were: background cpm, 621 t 142; positivecontrolcpm, 99,552 2 12,241; andethanolcontrol cpm, 93,162 t 16,211. Percent suppression was calculated as described in Figure 2.

FIGURE 9. Failure of AA to abrogate the GC-mediated antiproliferative effect. PBML were treated with ethanol (open triangle) or M DEX (closed triangle), reconstituted with AA at the indicated concentrations, stimulated with PHA-PMA, and cultured for 72 h at 3 7 T . Data points represent the mean of six individually performed experiments; control values were: background cpm, 928 ? 180; positive control cpm, 80,774 2 13,473; and ethanol control cpm, 84,188 ? 12,729. The percent responsewas calculated according to: [(test cpm - background cpm)/(control cpm - background cpm)l X 100%.


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PGE

LTB4

F I G U R E 10. AbrogationofDEX-induced suppression of eicosanoid production by anti-lipocortin I Ab. PGE, (left) and LTB, (right) levels

GCs exert their antiproliferative effects principally by directly targeting cytokine genes. The role of lipocortins in partially mediating the effects of GC remains to be determined.

Acknowledgments The authors thank Dr. Barbara P. Wallner and Dr. Jeoffrey Browning at Biogen (Cambridge, MA) for providing lipocortin 1 and 2 cDNA probes and anti-lipocortin I, 11, and V mAb. The expert technical assistance of Edward T. Hadro, Joumana W. Assi, and Dagmara M. Chudzik is greatly appreciated.

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DEX

IgC Cont. anti-LC

1 anti-LC II anti-LC 1/11

F I G U R E 11. Partial abrogation of DEX-mediated antiproliferative effects by anti-lipocortin Ab. PBML were stimulated with PHA-PMA and treated with ethanol (PHNPMA), 1O-’ M DEX (DEX), DEX plus control lgGl (IgG Cont.), DEX plus anti-lipocortin 1 Ab (anti-LC I), DEX plus anti-lipocortin II Ab (anti-LC II), or DEX plus anti-lipocortin I and antilipocortin II Abs (anti-LC 1/11), Data points represent the mean of eight individually performed experiments; control values were background cprn, 503 i- 120; positive control cprn, 82,502 ? 12,384; and ethanol control cpm, 80,774 IT 10,008. The percent response was calculated as described in Figure 9.

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PHA/PM4


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